Electrophoresis procedures are known for separating and analyzing proteins, nucleic acids and their reaction products based on differences in net surface charge, known as isoelectric focusing, and also based on differences in molecular size. Electrophoresis is a commonly used technique to separate proteins and other macromolecules (e.g. DNA and PAPA) based on the phenomenon that a molecule with a net charge will move in an electric field. The velocity v of migration of a protein (or any molecule) through a gel medium in the presence of an electric field is determined by the net charge z on the molecule, electric field strength E and frictional coefficient f. The general relationship may be stated as follows: EQU V=Ez/f
That is, the charged molecule will be propelled toward the oppositely charged field electrode by the electric field force (Ez). Its movement is opposed by viscous drag (fv) arising from friction between the moving molecule and the medium. The frictional coefficient depends on the viscosity of the medium and both the shape and mass of the molecule.
Isoelectric focusing relies on the property of certain bio-organic molecules, such as proteins and peptides, which have a three-dimensional structure on which ionizable surface groups (e.g., carboxyl, amino, imidazole, guanidinium) are clustered. These ionizable groups are amphoteric in nature and carry a net electrical charge, either positive or negative. At a unique gel pH value, called the isoelectric point, the net charge of surface groups is neutralized.
The isoelectric separation process is carried out by migration of the bio-organic molecules through a buffered gel substrate. A commonly used substrate is polyacrylamide gel. This gel consists of a mixture of acrylamide monomer and an appropriate cross-linking agent. The gel may be enclosed within a capillary tube or between parallel glass plates. The polyacrylamide gel is buffered with an ampholyte agent. Ampholyte consists of a mixture of low molecular weight amphoteric compounds having isoelectric points distributed over a predetermined range of pH values. The application of a constant-voltage, DC electric field across an ampholyte-buffered gel causes the ampholytes to shift and locate according to their specific leoelectric points, thereby establishing a pH gradient with sufficient buffering capacity and conductance for focusing the bio-organic molecules at their respective isoelectric points.
Isoelectric focusing has the effect of separating the bio-organic molecules with different electric charges into discrete bands along the length of the gel, with each band containing molecules having the same isoelectric point.
The bio-organic molecules may also be separated according to their different molecular weights by migration of the proteins through a gel slab which acts as a sieve. During migration through the gel, the different bio-organic molecules having different molecular weights produce separate clusters along the gel slab. Each cluster contains purified proteins having substantially the same molecular weight.
Electrophoretic separations are usually performed in gels since they function as molecular sieves that enhance separation. Molecules that are small compared to the pores in the gal move readily through the gel, while molecules larger than the pores are virtually immobile. The net result is that small proteins migrate further than large ones. Pore sizes of the gel can be controlled during gel production.
The gel slabs are usually made from polyacrylamide since it is chemically inert and readily formed by polymerization of acrylamide. Pore sizes are controlled by the concentration of acrylamide and methylenebisacrylamide (a cross-linking reagent) during polymerization. The gel slab is supported vertically and samples are placed on the exposed face of the gel slab. Current is applied such that the bottom of the gel slab is the anode; the negatively charged molecules migrate toward the bottom of the gel slab.
Protein bands are visualized by staining with silver or a dye such as Coomassie blue. Nucleic acid bands may be visualized with ultraviolet light and edthenium bromide staining. In addition, protein bands may be labeled with a radioactive tag for autoradiography or antibodies (Western blot), and nucleic acid bands may be labelled with antisense oligonucleotides (Northern and Southern blots).
Another separation technique which is useful for separating bio-organic molecules is known as high performance liquid chromatography. According to this technique, a mobile phase eluate, into which a bio-organic sample to be analyzed has been injected, is forced through a bed of micro-particulate chromatographic packing material at a predetermined linear velocity. The separation of components according to this technique depends on the eluate chemistry and the properties of the packing material utilized. Proteins and other bio-organic molecules may be separated by this procedure on the basis of molecular size, ionic properties, absorptive characteristics and hydrophobicity.